This application claims priority from Korean Patent Application No. 2003-62423 filed on Sep. 6, 2003, in the Korean Intellectual Property Office, the disclosures of which are incorporated herein in their entireties by reference.
1. Field of the Invention
The present invention relates to an L-threonine-producing Escherichia coli strain, and a method for producing L-threonine using the Escherichia coli strain.
2. Description of the Related Art
L-threonine is an essential amino acid and is widely used as a feed or food additive. In addition, L-threonine is used as a medical solution or a raw material for a drug synthesis.
L-threonine is produced by a fermentation process using a mutant strain derived from a wild-type strain of Escherichia coli (E.coli), Corynebacteria sp., Serratia sp., or Providencia sp. Examples of the mutant strain include an amino acid analogue- or drug-resistant mutant strain, and a diaminopimeric acid, a methionine, a lysine, or an isoleucine auxotrophic mutant strain that has an amino acid analogue- or drug-resistance. These mutant strains are disclosed in Japanese Patent Laid-Open Publication No. Hei. 2-219582; Appl. Microbiol. Biotechnol., 29. 550–553 (1988); Korean Patent Laid-Open Publication No. 1992-12423; and the like. Korean Patent Laid-Open Publication No. 1992-12423 discloses an L-threonine-producing E. coli TF4076 (KFCC 10718). The E. coli TF4076 is a methionine auxotrophic strain, and has a resistance to a threonine analogue (AHV: α-amino-β-hydroxy valeric acid), a lysine analogue (AEC: S-(2-aminoethyl)-L-cysteine), an isoleucine analogue (α-aminobutyric acid), and a methionine analogue (ethionine).
A fermentation process using a recombinant strain can also be used in production of L-threonine. For example, Japanese Patent Laid-Open Publication No. Hei. 5-10076 discloses a method for producing threonine in large scale using a recombinant strain of Serratia sp. containing a DNA fragment with genetic information of aspartokinase, homoserine kinase, homoserine dehydrogenase, and threonine synthase. In addition, a method of increasing the production of L-threonine using a gene derived from a strain of Providencia sp. resistant to antagonist of methionine metabolism is disclosed in Japanese Patent Laid-Open Publication No. Hei. 1-289493.
Meanwhile, the expression of a gene in a microorganism can be enhanced by increasing the copy number of the gene contained in the microorganism. For this, a plasmid that gives a greater copy number to a microorganism is used [Sambrook et al., Molecular Cloning, 2th, 1989, 1.3–1.5]. That is, the copy number of a plasmid in a microorganism is increased by inserting a target gene into the plasmid and then transforming the microorganism with the obtained recombinant plasmid. Attempts have been made to enhance the productivity of threonine using this method and a partial success was reported (U.S. Pat. No. 5,538,873). However, this technology using plasmid induces excessive expression of a specific gene in most cases, thereby imposing a heavy burden on a host microorganism. Furthermore, a plasmid loss during culture may be caused, thereby decreasing plasmid stability.
In order to solve these problems, addition of an antibiotic into a culture and using a plasmid containing an expression regulatory sequences were introduced by Sambrook et al. [Molecular Cloning, 2th, 1989, 1.5–1.6, 1.9–1.11]. In the case of using a plasmid containing an expression regulatory sequences, during the growth phase, a host microorganism is cultured so that no expression is induced, thereby decreasing a burden on the host microorganism. On the other hand, after the sufficient growth of the host microorganism, temporary expression is induced, thereby releasing a gene product. However, most of these plasmids containing an expression regulatory sequences can be used only when a final gene product is a protein. In a case where a gene product is a primary metabolite that is closely associated with the growth of microorganisms, expression of a target gene must be induced during the growth phase. Otherwise, it is difficult to anticipate the accumulation of the primary metabolite. Since threonine belongs to a primary metabolite, the above case is also applied to threonine.
As a method for producing threonine without these problems, inserting a threonine biosynthesis gene into chromosomal DNA is disclosed in U.S. Pat. No. 5,939,307. Further, there is disclosed a method for producing threonine by regulation of expression of threonine operon, for example, replacing a promoter of the threonine operon with tac promoter (WO 98/04715) or replacing an expression regulatory region of the threonine operon with cl repressor and PR promoter of E. coli λ phage (EP 0593792). In this case, however, since a gene on a chromosome is substituted by corresponding gene containing an inducible promoter, it is difficult to greatly increase the expression of threonine operon genes.
In this regard, while searching for solutions to the problems of the above-described conventional L-threonine production methods, considering the fact that metJ gene represses the expression of metL gene, one of genes regulating the biosynthesis pathway of threonine and methionine, the present inventors found that an E. coli strain containing chromosomal DNA with inactivated metJ gene can produce a high yield of L-threonine, and then completed the present invention.